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Regulation of cytochrome P450 and P450 mRNA in human granulosa-luteal cells Ge, Hong


This thesis investigated the expression and regulation of cytochrome P450s, aromatase (P450arom) and cholesterol side chain cleavage (P450scc) enzyme mRNAs in human granulosa-luteal cells using reverse transcription-polymerase chain reaction (RT-PCR). The two enzymes are instrumental in steroid hormone biosynthesis. The steroid hormone biosynthesis by the ovary is determined in part by the pattern of gonadotropin secretion and also appears to be under the influence of local regulators which provide the fine tuning at a local level in a gonadotropin dependent or independent way. In this study, the granulosa-luteal cells were aspirated from preovulatory follicles obtained from women undergoing in vitro fertilization. A highly sensitive method, reverse transcription-polymerase chain reaction (RT-PCR), was used to quantitate P450arom and P450scc mRNA expression. Two sets of primers designed from cDNA sequences were used to amplify cDNAs from granulosa-luteal cells. The authenticity of the PCR products was confirmed by Southern blot hybridization with specific cDNA probes. It was shown that cAMP and hCG increased the levels of P450arom and P450scc mRNA by 2-3 fold and 4-5 fold. GnRH at 10⁻⁶ M had no significant effects either on basal or on hCG or cAMP stimulated P450arom and P450scc mRNA expression. Under the same cell culture conditions, GnRH at lower concentrations 10⁻⁸ or 10⁻⁹ M did not show significant effects on basal P450arom and P450scc gene expression, but significantly inhibited hCG-stimulated P450arom and P450scc gene expression and progesterone production. By contrast, GnRH treatment had no significant effect on exogenous 8-br-cAMP-stimulated P450arom and P450scc gene expression. Studies on the time course for the inhibitory effect indicated that GnRH at 10⁻⁶ M significantly inhibited hCG stimulated P450arom and P450scc mRNA expression from 24 to 48 hour of treatment. These results provide evidence that chorionic gonadotropin stimulates P450arom and P450scc mRNA expression and cAMP could be a mediator for a positive regulation. GnRH not only plays a role in neuroendocrine regulation, but also has local effects on extrapituitary sites. In human ovary, the paracrine/autocrine effects of GnRH on steroidogenesis of human granulosa-luteal cells may play an important role in determining an individual follicles's response to gonadotropins.

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